The overall goal of this project is to elucidate the effects that local structural perturbations in DNA have on an advancing RNA polymerase (RNAP) as it transcribes DNA to form an RNA product. Transcription is the first step in the expression of genetic information in DNA, and as such its regulation is central to the regulation of gene expression. During RNA chain elongation, smooth and rapid progress of the ternary complex, consisting of RNAP, its DNA template, and nascent RNA product, can be hampered intrinsically by a variety of sequence and/or structural features of the DNA template or RNA product, and regulated by a variety of extrinsic factors that associate with RNAP. Defects in elongation regulatory mechanisms have been associated with many human diseases, and therefore the factors involved in this process are important preventive and therapeutic targets. To understand the regulation of transcriptional elongation, we must first understand the interactions between RNAP and its DNA template and RNA product that govern elongation. It is clear that interactions between RNAP and downstream DNA can play significant roles in all aspects of progress of the transcription complex along the DNA, from promoter escape to pausing and termination. However, the mechanism(s) underlying these roles are unknown, as are the ways in which extrinsic regulatory proteins affect these roles. Observations (including my own preliminary studies) of effects of downstream DNA sequences on elongation have included examples of pausing and arrest upstream of A-tracts or T-tracts, and it has been speculated that these effects may be due to A-tract- (or T-tract-) induced DNA bending. No systematic study of the effects of such downstream DNA structures on transcriptional elongation has been undertaken. I propose to use a series of transcription templates containing a systematically varied site of bending to explore the relationship between DNA bending and RNA chain elongation kinetics in transcription by E. coli RNAP. Using primarily an in vitro biochemical approach employing these templates, mutant forms of RNAP known to be altered in their elongation properties, as well as extrinsic regulatory proteins known to affect the elongation process, I plan to elucidate the mechanism(s) by which DNA bending affects elongation.